A. COAGULATION
Coagulation of blood occurs by either the "intrinsic pathway" or the "extrinsic pathway", whereby certain blood proteins interact in a cascade of proteolytic activations to ultimately convert soluble fibrinogen to insoluble fibrin. These threads of fibrin are cross-linked to form the scaffolding of a clot; without fibrin formation, coagulation cannot occur.
The intrinsic pathway consists of seven steps: (1) the proteolytic activation of Factor XII; (2) activated Factor XII cleaves Factor XI to activate it; (3) activated Factor XI cleaves Factor IX, thereby activating it; (4) activated Factor IX interacts with activated Factor VIII to cleave and activate Factor X; (5) activated Factor X binds to activated Factor V on a membrane surface, which complex proteolytically cleaves prothrombin to form thrombin; (6) thrombin proteolytically cleaves fibrinogen to form fibrin; (7) fibrin monomers assemble into fibrils, which are then cross-linked by Factor XIII.
The extrinsic pathway consists of the following steps: (1) upon rupture of a blood vessel, Factor VII binds to tissue factor, a lipoprotein present in tissues outside the vascular system; (2) Factor VII is activated to Factor VIIa by proteolytic cleavage; and (3) the Factor VIIa-tissue factor complex cleaves and activates Factor X. Thereafter, the extrinsic pathway is identical to the intrinsic pathway, i.e. the two pathways share the last three steps described above.
One of the plasma proteins, coagulation Factor IX ("CFIX") is synthesized in the liver by hepatocytes as a 415 amino-acid polypeptide and then post-translationally modified to a glycoprotein of molecular weight 56,000 Daltons by a carboxylase requiring vitamin K as a cofactor. CFIX is thus one of the group of "vitamin K-dependent" plasma proteins.
Factor VII is another vitamin K-dependent clotting protein that is similar to CFIX in size and structure.
Factor VIII, a non-vitamin K-dependent protein, is a much larger protein, with a molecular wieght of near 300,000 daltons (300 kDa). It is activated by thrombin, which cleaves the molecule in several places to form Factor VIlla (the activated form). In plasma, Factor VIII binds to von Willebrand Factor (vWF) and circulates as complexes with vWF, which stabilizes the labile Factor VIII molecule.
Disturbing the balance of the cascade involved in the intrinsic pathway results in various coagulation disorders. The absence or reduction of an intrinsic Factor X-activating moiety (a "tenase") at step (4) results in the defective-coagulation condition known as hemophilia. Hemophilia A, the most common, results from a mutation in the gene for Factor VIII; Hemophilia B, also known as Christmas Disease, results from a mutation in the gene for Factor IX. Hemophilia B, like Hemophilia A, is X-linked and accounts for approximately 12% of hemophilia cases. The symptoms are identical to those of Hemophilia A: excessive bleeding upon injury; and spontaneous bleeding, especially into weight-bearing joints, soft tissues, and mucous membranes. Repeated bleeding into joints results in hemarthroses, causing painful crippling arthropathy that often necessitates joint replacement. Hematomas in soft tissues can result in pseudo tumors composed of necrotic coagulated blood; they can obstruct, compress, or rupture into adjacent organs and can lead to infection. Once formed the hematomas are difficult to treat, even with surgery. Recovery of nerves after compression is poor, resulting in palsy. Those bleeding episodes that involve the gastrointestinal tract, central nervous system, or airway/retroperitoneal space can lead to death if not detected. Intracranial bleeding is a major cause of death in hemophiliacs.
Current treatment of these symptoms consists of intravenous replacement therapy with Factor VIII or Factor IX concentrates. Treatment of major bleeding episodes is by bolus injection of concentrate. As described above, however, tissue damage remains even after prompt detection and treatment. Prophylactic treatment is recommended to prevent this pain and debilitation. Upon injection, 50% of Factor IX is immediately bound to vascular endothelial cells and/or diffuses into the extravascular space. The remaining 50% has a half life in circulation of approximately 24 hours. These infusion kinetics result in the need for injections once to twice per week or more to maintain minimal therapeutic levels in the plasma. While this regimen is inconvenient and stressful for the patient, it is also not totally effective. Progressive, cumulative tissue damage continues with each bleeding episode prior to the onset of treatment.
B. VITAMIN K-DEPENDENT PLASMA PROTEINS
The group that comprises the vitamin K-dependent plasma proteins consists to date of Factor II, Factor VII, Factor IX, Factor X, Protein C, Protein S, and Protein Z. These proteins exhibit significant homology on all levels: gene organization, amino acid sequence (primary structure), protein folding (secondary structure), post-translational modifications, activation, and function (Hedner and Davie, Chapter 84: Introduction to Hemostasis and the Vitamin K-Dependent Coagulation Factors, in C. R. Scriver et al. (Eds.), Metabolic Basis of Inherited Disease, 6th edition, McGraw-Hill, New York N.Y., (1989), pp. 2107-2134). A comparison of the features of the six- well-characterized vitamin K-dependent plasma proteins follows (Protein Z is excluded due to a paucity of information regarding its properties and function):
__________________________________________________________________________ Feature Factor II Factor VII Factor IX Factor X Protein C Protein S __________________________________________________________________________ No. gla domains 10 10 12 11 9 11 No. EGF's (w/.beta.) 2 Kringle 2 2 2 2 4 .beta.'s Activation cleavage 2 by Xa 1 by Xa 2 by XIa 2 by IXa 1 by IIa -- Function serine serine serine serine serine cofactor, protease protease protease protease protease Protein C Catalytic domain His43 His41 His41 His42 His42 -- Asp99 Asp90 Asp89 Asp88 Asp88 Ser205 Ser192 Ser185 Ser185 Ser191 No. carbohydrates 3 3 2 2 4 3 __________________________________________________________________________
The "gla" domains consist of the first 40 to 45 amino acid residues with multiple .gamma.-carboxyglutamic acid residues that are formed by the carboxylation of the amino acid glutamic acid by a membrane-bound complex requiring vitamin K. They are required for the calcium-dependent binding of the protein to phospholipid surfaces.
The "EGF" domains are composed of 40 to 50 amino acids that show considerable sequence similarity to Epidermal Growth Factor (EGF) and its precursor. The first EGF domain in each of these proteins contains a .beta.-hydroxyaspartic acid modification. While Protein S does not contain these EGF domains, it does contain three .beta.-hydroxyaspartic acid residues and one .beta.-hydroxyasparagine residue. Factor II, also known as prothrombin, contains 2 Kringle regions in the place of these EGF domains. These Kringle domains are also found in Factor XII, Plasminogen, Tissue Plasminogen Activator, and Urokinase, which while not vitamin K-dependent proteins, are plasma proteins with proteolytic activity that are involved in coagulation.
Five of the six vitamin K-dependent plasma proteins are activated by proteolytic cleavage by the preceding member of the coagulation cascade. Three have two cleavages, releasing an activation peptide that shielded the catalytic domain. Activation of Factor X by Factor IXa involves a single cleavage that releases an activation peptide from the heavy chain of this two-chain protein. Factor VII is activated by a single cleavage of the single chain of the polypeptide, without the release of an activation peptide.
Five of the six vitamin K-dependent plasma proteins are serine proteases upon activation. These proteases have histidine, aspartic acid, and serine residues in analogous positions within their catalytic domains. Protein S is a cofactor for Protein C and does not in itself have a catalytic domain known at this time.
The six vitamin K-dependent plasma proteins are glycoproteins with two to four N-linked glycosylation sites in the EGF and catalytic domains and/or activation peptides.
The vitamin K-dependent plasma proteins are similar enough in characteristics to co-purify throughout most of their purification procedures. All six vitamin K-dependent plasma proteins are found in the same fractions throughout most steps of both commonly used purification procedures. The barium citrate or aluminum hydroxide adsorptions of Cohn fractions co-purify Factors II, VII, IX, and X as well as Protein C; anion exchange chromatography on resins such as DEAE-Sephadex or DEAE-Sepharose co-purifies Factors II, IX, and X along with trace amounts of Factor VII and Protein C, from cryo-poor plasma.
C. CURRENT FACTOR IX PREPARATIONS
The two manufacturers that have Factor IX concentrates on the U.S. market provide them in lyophilized form. Armour Pharmaceuticals (now Centeon) produces Mononine, which, upon reconstitution with sterile water for injection (WFI), is delivered in a composition of: 0.01 moles/liter histidine, pH 7.05; 0.066 moles/liter sodium chloride; 3% mannitol. AlphaNine SD is manufactured by Alpha Therapeutics and upon reconstitution with sterile WFI is delivered in a composition that includes: 0.04 units heparin/unit FIX; 1 milligram dextrose/unit FIX. One lot of Mononine was found to have an in vitro half life when reconstituted of 13 days at 37.degree. C. To avoid repeated invasive treatments as is found with the current therapies for prophylaxis, stabilities of at least 30 days at 37.degree. C. and at least 365 days at 4.degree. C. are necessary.
D. CURRENT FACTOR VIII PREPARATIONS
Three manufacturers produce affinity-purified plasma Factor VIII concentrates for the U.S. market, all of which are lyophillized products. Baxter Healthcare/Hyland Division manufactures two products by the same method: Antihemophilic Factor (Human), Method M, Monoclonal Purified (AHF-M) is produced for the American Red Cross (ARC) from volunteer donor plasma collected by the ARC; and Hemophil M is produced from commercial plasmapheresis plasma. These products, upon reconstitution with sterile WFI, are delivered in a composition of 12.5 mg/mL human albumin, 1.5 mg/mL PEG, 0.030 M glycine, and 0.055 M histidine. Armour/Centeon produces Monoclate, which upon reconstitution with sterile WFI is delivered in a composition of 10-20 mg/mL human albumin, 0.30-0.45 M sodium chloride, 2-5 mM calcium chloride, 0.8% mannitol and 1.2 mM histidine. Alphanate, manufactured by Alpha Therapeutic Corporation, upon reconsitution with sterile WFI is delivered in a composition of 0.5-10 mg/mL human albumin and not more than 10 mM calcium, 2 .mu./mL heparing, 0.055 M histidine and 0.3 M arginine.
In addition, two freeze-dried recombinant Factor VIII products are presently on the market. Recombinate is produced by Baxter HealthCare and Kogenate is produced by Bayer Corporation.
E. OTHER STABLE AQUEOUS PLASMA PROTEIN PREPARATIONS
The components of fibrin sealant, or fibrin glue, have been formulated in liquid that results in an activity half life of greater than 6 months at 4.degree. C. for both the thrombin and fibrinogen concentrate components (Chabbat et al., Thrombos. Res. 76:525-533 (1994)). The thrombin component is in a formulation of: arginine, 1.6 millimoles/liter; benzamidine less than 1 milligram/liter; gluconate, 1.7 millimoles/liter; calcium, 22 millimoles/liter; pH 6.6. The fibrinogen concentrate component is in a formulation of: aprotinin, 250 Kiu/milliliter; glycine, 1.2 grams/liter; ethanol, less than 0.1 grams/liter; pH 7.6.
F. NON-AQUEOUS LIQUID PROTEIN FORMULATIONS
There are very few precedents for the formulation of pharmaceutically significant proteins in non-aqueous liquid formulations. However, the components of Fibrin Sealant, fibrinogen and thrombin (activated Factor II), have been formulated in a non-aqueous ethanol solution to facilitate storage and delivery of these components in a single delivery unit without premature activation.
G. DELIVERY OF PHARMACEUTICAL PREPARATIONS BY CONTINUOUS INFUSION
Pumps have been in use for the continuous delivery of pharmaceuticals in liquid formulation. External syringe-delivery pumps are used in the U.S. and abroad for the delivery of insulin, antibiotics, chemotherapeutics, and hormones. These pumps can deliver liquids in programmed continuous doses or in bolus injections, as necessary, for intravenous, subcutaneous, or intraperitoneal delivery. Reservoir capacity ranges from 1 milliliter to 1500 milliliters. The pumps are powered by batteries with a 2 to 3 month life. It is therefore desirable for the liquid Factor IX formulation to have a stability of at least 30 days at 37.degree. C., requiring physician oversight once per month instead of twice per week as for the current prophylactic therapy. Externally worn pumps of the types described have been used to deliver Factor VIII and Factor IX to hemophilia patients for short-term perisurgical prophylaxis to prevent excessive bleeding that could otherwise occur with invasive procedures.
In addition, two implantable pumps are available for use in humans, but have not been employed for delivering coagulation factors to hemophilia patients. These pumps are designed to be surgically implanted in the chest wall or abdomen, cushioned by subcutaneous fat, with the exit catheter anchored for peritoneal, hepatic artery or perispinal access. The pumps are filled externally by injection through the skin. The Arrow Model 3000 Implantable Pump (Arrow Therix, Walpole, Mass.) is licensed for clinical use and is applied primarily for the delivery of pain medications and chemotherapeutic agents for liver cancer. It operates by a titanium bellows with a capacity of 30 mL and can be obtained in three pre-set flow rates: 0.5, 1.0 and 2.0 mL/day, with special hypodermic needles for bolus infusion. The MiniMed 2001 (MiniMed Technologies, Sylmar, Calif.) is awaiting licensure for use in delivery of insulin. This peristatic pump has a titanium reservoir with a capacity of 15-18 mL and can be externally controlled by a programmable communicator, allowing for variable flow rates and bolus override when required.
Hydrogels, particularly chitin hydrogels or chitosan hydrogels, have previously been used for sustained drug release (Chandy and Sharma, Biomat. Art. Cells & Immob. Biotech. 19:745-760 (1991)). More recently, in vivo release of Factor IX from a subcutaneous injection of a negatively charged derivative of chitosan, N,O-carboxymethyl chitosan (NOCC), that had Factor IX incorporated within it, demonstrated that slow diffusional release from the hydrogel resulted in the appearance of the Factor IX in the plasma and at lower peak levels that more closely approximated normal levels than did direct subcutaneous injection of Factor IX without the hydrogel; it also resulted in longer delivery of Factor IX at clinically significant levels (Oral presentation at XVth Congress of the International Society on Thrombosis and Haemostasis, Jerusalem, Israel on Jun. 11, 1995).